This is the U.S. national stage application under 35 U.S.C. 371 of PCT Application No. PCT/JP97/00665, filed Mar. 5, 1997.
The present invention relates to an amino acid sequence of membrane protein occludin in a tight junction (hereinafter referred to as xe2x80x9cthe TJxe2x80x9d) of a human, a dog and a mouse, and a DNA for encoding the amino acid sequence.
In multicellular animals, the information on cellular adhesion between adjacent cells is deeply concerned with the regulation and maintenance of vital phenomena such as cellular proliferation and differentiation, inflammation and cancer metastasis. Intercellular adhesion molecules which take part in the adhesion of the cells frequently assemble together on the surfaces of these cells to form a specifically differentiated membrane region for the adhesion. Particularly, intercellular adhesion molecules such as cadherins are known to be firmly bound to cytoskeleton in the cytoplasmic domain of epithelial cells. Such membrane regions are called intercellular adhesion apparatus and chiefly classified into the following four structures: gap junction (GJ), adherens junction (AJ), desmosome and tight junction (TJ).
These adhesion apparatus has first been identified under an electron microscope, and as a result of the investigation and research of constituent proteins, the importance of their physiological and pathological significance has become the focus of increased interest. The proteins that are called the so-called adhesion molecules specifically exist in these adhesion apparatus, and the adhesion molecules of the AJ are cadherins and various kinds of cadherins such as N-cadherin and P-cadherin have been identified so far [Takeichi, M. et al., xe2x80x9cSciencesxe2x80x9d, Vol. 251, p. 1451-1455, (1991)]. As adhesion molecules of the desmosome, desmoglein and desmocollin are known, and according to recent studies, it has been elucidated that their structures are similar to those of the cadherins [Buxton, R. S. et al., xe2x80x9cJ. Cell Biol.xe2x80x9d, Vol. 121, p. 481-484, (1993)]. The adhesion molecules of the GJ are called connexin, and it is known that connexin holds transmembrane domains at four different sites and both of its N-terminal and C-terminal protrude on the cytoplasmic side of the membrane.
The TJ is an intercellular adhesion apparatus peculiar to epithelial cells and endothelial cells, where the cell membranes of contiguous cells are seen completely tightly apposed. The TJ surrounds individual cells and functions as a barrier to block or regulate permeation of water-soluble molecules between the luminal and basement membrane sides of a cell layer. It has also been described to act as a fence partitioning the cell membrane into apical and basolateral sides in order to maintain the polar distribution of such membrane proteins as ion channels and pumps as well as lipids in the cell membrane [Schneeberger, E. E. et al., xe2x80x9cAm. J. Physiol.xe2x80x9d, Vol. 262, P. L647-L661, (1992)]. Owing to these functions of the TJ, milieus consisting of different fluid compositions are formed on the opposite sides of a cell layer, so that the polarity of the cell layer is maintained; hence the TJ can be said to be a fundamental structure of vital importance to multicellular organisms.
However, analysis of the molecular structure of the TJ has been less progressing, compared to other adhesion apparatus. In fact, it has constituted a serious drawback to the pursuit of molecular biological research on the TJ that the TJ adhesion molecule itself has not been identified yet.
The present inventors have established a method for isolation of AJ from rat liver, and have identified many proteins such as radixin and ZO-1 from this isolated AJ [Tsukita, Sh. et al., xe2x80x9cCurr. Opin. Cell Biol.xe2x80x9d, Vol. 4, P. 834-839, (1992)]. From research on ZO-1 and histologic findings for the AJ and the TJ, it can be presumed that the proteins in the AJ also contain a protein of the TJ. In view of this, the present inventors have isolated AJ from chick liver, prepared a monoclonal antibody against the AJ as the antigen, and carried out structural analysis of the TJ-constituting protein using the antibody specifically reacting with the TJ. As a result, the present inventor has been successful in the structural analysis of a novel constituent protein dissimilar to known proteins, and designated the protein as occludin [Furuse, M. et al., xe2x80x9cJ. Cell Biol.xe2x80x9d, Vol. 123, p. 1777-1788, (1993)].
This chick occludin is a 56 KDa protein composed of 504 amino acids, characterized conspicuously by transmembrane domains at four sites in the half of its N-terminus, with both the N- and C-terminals facing the cytoplasm and with two extracelluar loops.
From subsequent studies, occludin was inferred to be an important factor in the analysis of physiological function of the TJ at the cellular level as well as at the whole body level, and drew much attention of investigators.
No further study has progressed, nevertheless, since the said protein has its origin in the chicken species greatly remote from humans. Thus, structural analysis of occludin of human origin has been expected for the sake of elucidation of the physiological function and medical analysis of the TJ. There is as yet no report of success in the elucidation of human occludin despite worldwide competition in research for this purpose in this field.
It is accordingly an object of the present invention to provide amino acid sequences of human, canine and mouse occludins and the DNAs encoding them.
In their report on the gene for human neuronal apoptosis inhibitory protein (NAIP), Roy et al. documented occurrence of a DNA fragment possessing a base sequence analogous to the C-terminal region of chick occludin in NAIP gene deletion mutants [Roy, N. et al., xe2x80x9cCellxe2x80x9d, Vol. 80, p. 167-178, (1995)]. To ascertain whether the said sequence actually encoded a part of the human analogue of occludin or not, the present inventor selected primers out of the base sequence analogous to that of chick occludin and made a scrupulous screening with a cDNA library of human intestinal epithelial cell strain T84 as a template for PCR. The present inventor has thus succeeded in the analysis of the whole structure of human occludin. Further, the inventor has completed analyses of mouse and canine occludins, prepared anti-occludin monoclonal antibodies, and verified with histologic staining that the occludins were transmembrane type TJ proteins.